Our work during the previous funding period established the existence of a mechanism that produces extracellular adenosine, i.e., the extracellular 3',5'-cAMP-adenosine pathway that involves four sequential steps: 1) intracellular production of 3',5'-cAMP by adenylyl cyclases;2) efflux of intracellular 3',5'-cAMP to the cell surface mediated by multidrug resistance proteins (MRPs);3) extracellular metabolism of 3',5'-cAMP to 5'- AMP by ecto-3',5'-cAMP-phosphodiesterases;and 4) extracellular conversion of 5'-AMP to adenosine by CD73. This pathway is gaining recognition as an important mechanism for adenosine biosynthesis in many organ systems, including the kidney and the cardiovascular system. Recently, we conceived the hypothesis that there may exist, in addition to the extracellular 3',5'-cAMP- adenosine pathway, an extracellular 2',3'-cAMP-adenosine pathway: mRNA turnover-intracellular 2',3'- cAMP-extracellular 2',3'-cAMP-extracellular 2'-AMP/3'-AMP-extracellular adenosine. The rationale for this hypothesis is: 1) mRNA turnover involves ribonucleases that cleave the phosphodiester bonds in the polyadenine tail of mRNA forming 2',3'-cAMP;2) Some MRPs rapidly transport cyclic nucleotides into the extracellular space;and 3) Enzymes exist that could serve as ecto-2',3'-cAMP-phosphodiesterases and ecto- 2'/3'-nucleotidases to hydrolyze extracellular 2',3'-cAMP to 2'-AMP/3'-AMP and extracellular 2'-AMP/3'-AMP to adenosine, respectively. This pathway could be extremely important in producing extracellular adenosine whenever cells are exposed to stressful stimuli that enhance mRNA turnover, thus providing the "retaliatory" metabolite, adenosine, to mitigate cellular damage. To test this hypothesis, we recently established an innovative technology in our lab that allows us to measure purine metabolites with extreme accuracy, specificity and sensitivity, i.e., the Thermo Electron TSQ Quantum-Ultra liquid chromatograph-mass spectrometer. Using this instrument, we made fascinating preliminary observations in intact kidneys and in cultured preglomerular vascular smooth muscle cells and mesangial cells supporting the concept that the kidney produces more extracellular 2',3'-cAMP than 3',5'- cAMP and that this 2',3-cAMP is metabolized to adenosine. These are indeed potentially transformative preliminary observations! Accordingly, the overall goal of this competing renewal application is to determine whether the renal extracellular 2',3'-cAMP-adenosine pathway exists (Specific Aims 1 and 2), whether it is mediated by ecto-enzymes that are different from those mediating the 3',5'-cAMP- adenosine pathway (Specific Aim 3) and whether the 2',3'-cAMP-adenosine pathway has the potential to cause biological effects (Specific Aim 4). PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand how the body makes a chemical called adenosine. Adenosine is a naturally occurring chemical that affects every organ system in the body. Therefore, knowledge regarding how the body makes adenosine is important for elucidating mechanisms of many diseases including diseases of the kidney, heart, blood vessels and brain, as well as inflammatory diseases and cancer. Adenosine is also an important pharmaceutical (drug) that has multiple uses, for example to treat cardiac arrhythmias, to diagnosis coronary artery disease and to treat myocardial ischemia/reperfusion injury. This project will examine the metabolism of another naturally occurring chemical called 2',3'-cAMP to adenosine. It is likely that 2',3'-cAMP is an "adenosine prodrug" that could have multiple uses in clinical medicine as a safer form of adenosine. Rapamycin (sirolimus) is a drug used in drug-eluting coronary stents to prevent a process called restenosis. Interestingly, a major aspect of the pharmacology of rapamycin is that it may activate the adenosine-producing system that is the subject of this project. It is conceivable that this project will provide information leading to safer and more effective drug-eluting stents.